As engineered antibody fragments with improved pharmacokinetic properties for tumor targeting are developed and validated in small animal models, they need to be tested in pilot human imaging studies. The use of positron emission tomography (PET) potentially provides for a high resolution, high sensitivity imaging approach as compared to conventional gamma camera/single photon emission computed tomography (SPECT) imaging. The use of PET and positron labeled engineered antibody fragments may be a highly sensitive and specific approach for tumor receptor imaging. In this project we will translate the best antibody labeled fragment (minibody or diabody) targeted against carcinoembryonic antigen (CEA) and labeled with 123I (for SPECT imaging) and 124I (for PET imaging) in a pilot clinical trial of 10 patients with rising CEA levels and suspicion for tumor recurrence. We will then directly compare 18F-fluorodeoxyglucose (FDG) PET to 124I-antibody fragment in patients with suspected recurrence for colorectal cancer and rising CEA levels scheduled for surgery. This multicenter trial will be performed at UCLA, City of Hope, and Memorial Sloan-Kettering Cancer Center (MSKCC). This trial will allow us to directly compare the best PET tracer currently available (FDG) to a new generation of tracer in terms of potential gains in sensitivity and specificity. Furthermore, in order to study a different receptor system for eventual use with PET, we will image prostate cancer patients with a high suspicion for metastases utilizing an 111In-anti-prostate stem cell antigen (PSCA) antibody and SPECT. This work will set the foundation for eventually pursuing prostate cancer receptor based imaging with engineered antibody fragments coupled to positron emitting isotopes. Finally, we will also image a 64Cu-anti-CEA antibody fragment (after its imaging characteristics have been optimized in animal studies) in patients with elevated CEA levels and suspicion of recurrence in order to study potential advantages of 64Cu vs. 124I when imaging with PET and antibody fragments. This project will help to combine expertise in PET imaging at UCLA and MSKCC with expertise in engineering novel antibody fragments at City of Hope in order to significantly advance the field of cancer imaging. Through a systematic translation from animal studies to pilot clinical studies to a multicenter trial, this work should help set the way for further utilization of engineered antibody fragments for improved cancer diagnosis and potentially better patient management.